Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

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AquaLifefu
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Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#1 Post by AquaLifefu » Mon Apr 04, 2022 8:04 am

I'm currently in the process of making a permanent mount and found a video in the community that said to use xylene as clearing agent. I was told it was toxic but didn't think it was that toxic until I done research about it "toxic include acute neurotoxicity, cardiac and kidney injury, cancer, blood dyscrasias, skin diseases, gastrointestinal disturbances, musculoskeletal system disorders, fetotoxicity".

I'm too clumsy and absent minded so I done some research and came across two research paper.

"Vegetable Oils as Clearing Agents"
https://www.sciencedirect.com/science/a ... 2015300328

"Comparing the efficacy of coconut oil and xylene as a clearing agent in the histopathology laboratory" https://www.ncbi.nlm.nih.gov/pmc/articl ... po=19.1176

From what I read on both of them, the conclusion is that there are no different and coconut oil(vegetables oil) should be use as alternative rather than xylene, have anyone try this out yet? I read through a lot of permanent mount instructions and none ever suggest to use any type of vegetable oil.

" Conclusion:
Coconut oil may be substituted for the highly hazardous xylene as a clearing agent without compromising the quality of histological details."

"Conclusion
This study has shown that palm kernel oil, cococnut oil and groundnut oil successfully cleared the wood sections processed in the vegetable oils as did by xylene and clove oil. During handling, the oils were not hazardous, except for the mild rancid smell from PKO that did not constitute any hindrance during clearing. No instance of feature distortion, inadequate translucency or threat to human health was experienced during processing, therefore the use of these oils as alternatives to clove oil and xylene will definitely reduce the cost slide preparation since the vegetable oils used in this study were far cheaper than the conventional clearants."

" The only drawback associated with coconut oil, is its tendency to get solidified at a lower temperature. However, this can be overcome by performing the clearing procedure in an incubator, maintaining the required temperature"

Honey and olive oil to subtitute formalin and xylene.https://pubmed.ncbi.nlm.nih.gov/2872199 ... %20xylene.
Last edited by AquaLifefu on Mon Apr 04, 2022 9:07 am, edited 1 time in total.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#2 Post by AquaLifefu » Mon Apr 04, 2022 8:46 am

Both palm kernel oil and coconut oil solidifies at room temperature and require an incubator to keep it liquid for clearing, so the most convenient oil to use would be ground nut oil(peanut oil) from what I understand . https://www.srmjrds.in/article.asp?issn ... avanakumar

They also talk on why xlyene are still use nowadays due to unwillingness to change.

"Even though hefty amount of clearing agents are present, anatomists and pathologists have preferred to use “xylene” over the past century. “xylene” offers several advantages: it is a stabilized liquid, effectively removes the dehydration agent, effectively removes the molten wax, creates significant harm to the body and minimal fragility, and is cost-effective.[4] In addition, pathologists are taught to look at parts that have been cleared with “xylene” and therefore are unwilling to alter the microscopic look of diagnostic tissues using a separate clearing agent. Although several alternative chemicals such as limonene reagents, aliphatic and aromatic hydrocarbons, and mineral oil mixtures are used to replace “xylene” during tissue processing as a clearing agent.”
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#3 Post by Alexander » Mon Apr 04, 2022 9:20 am

Rather than those fancy vegetable oils I recommend the use methyl benzoate instead of xylene. One of its advantages is its tolerance against traces of water. It is not poisonous but more expensive than xylene.

When it comes to the "horrible" danger xylene represents, please remember that your mounting media still contains xylene just as many glues available in the local hardware store do.

Xylene is not that dangerous if handled properly following laboratory safety rules. Generations of pathologists survived the use of xylene.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#4 Post by AquaLifefu » Mon Apr 04, 2022 9:39 am

Alexander wrote:
Mon Apr 04, 2022 9:20 am
Rather than those fancy vegetable oils I recommend the use methyl benzoate instead of xylene. One of its advantages is its tolerance against traces of water. It is not poisonous but more expensive than xylene.

When it comes to the "horrible" danger xylene represents, please remember that your mounting media still contains xylene just as many glues available in the local hardware store do.

Xylene is not that dangerous if handled properly following laboratory safety rules. Generations of pathologists survived the use of xylene.
Not sure if fancy vegetables oil was suppose to be sarcastic to be honest since they could be pick up in any grocery store. Contain xylene isn't the same as having a container of the chemical laying around potentially spilling it, or worse, people who may neglected safety or not know about it. I have a mask with charcoal filter, wore glove, do my danger chemicals outside but I am just a hobbyists who is interested in microscopy ,if there is a safer subtitute with no real downside, I don't see why we couldn't suggest other to use that alternative instead.

It would give other people who may be interested in getting into making their own permanent slide without the intimidating of dangerous chemical and the safety aspect that come with it, plus it is a lot better for the environment if I happen to spill vegetable oil rather than xylene.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#5 Post by Hobbyst46 » Mon Apr 04, 2022 10:24 am

AquaLifefu wrote:
Mon Apr 04, 2022 9:39 am
Alexander wrote:
Mon Apr 04, 2022 9:20 am
Rather than those fancy vegetable oils I recommend the use methyl benzoate instead of xylene. One of its advantages is its tolerance against traces of water. It is not poisonous but more expensive than xylene.

When it comes to the "horrible" danger xylene represents, please remember that your mounting media still contains xylene just as many glues available in the local hardware store do.

Xylene is not that dangerous if handled properly following laboratory safety rules. Generations of pathologists survived the use of xylene.
Not sure if fancy vegetables oil was suppose to be sarcastic to be honest since they could be pick up in any grocery store. Contain xylene isn't the same as having a container of the chemical laying around potentially spilling it, or worse, people who may neglected safety or not know about it. I have a mask with charcoal filter, wore glove, do my danger chemicals outside but I am just a hobbyists who is interested in microscopy ,if there is a safer subtitute with no real downside, I don't see why we couldn't suggest other to use that alternative instead.

It would give other people who may be interested in getting into making their own permanent slide without the intimidating of dangerous chemical and the safety aspect that come with it, plus it is a lot better for the environment if I happen to spill vegetable oil rather than xylene.
The chemistry of the effect of formalin on tissues is not the same as the chemistry of the effect of alcohols, honey or oils. Formalin has been used for many generations. yes, it is toxic, carcinogenic and more. Yes, its smell is disgusting. Yes, it should be definitely avoided in home labs, where safety measures and facilities are limited. Xylene is hazardous though less than formalin. But for some purposes there are no replacements for the real stuff.

Moreover, The authors of the paper in the link mention that their results are in contradistinction to the results of another research group.

So, I would suggest:
1. Check the literature to find out if anyone else has tried the honey and oil for that purpose, perhaps there is an established conclusion by several researchers if the method works. Or find citations of that article in later literature.
2. Check out Walter Dioni's publications on alternative, home-safe protocols for specimen preparation.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#6 Post by Microscopy_is_fun » Mon Apr 04, 2022 11:07 am

AquaLifefu wrote:
Mon Apr 04, 2022 9:39 am
It would give other people who may be interested in getting into making their own permanent slide without the intimidating of dangerous chemical and the safety aspect that come with it, plus it is a lot better for the environment if I happen to spill vegetable oil rather than xylene.
I think the easiest for you will be to use a different mounting medium which doesn't require xylene&Co for clearing. You could e.g. use Euparal, which only needs ethanol and isopropanol prior to mounting. Morphisto's safeline products provide chemicals which are even more environmentally friendly and "kitchen-compatible" (although at the cost of some performance):

https://www.morphisto.de/en/shop/list/f ... dukte/176/

For the wood sections mentioned in the paper which you referenced, I do not see the necessity to use xylene or hot vegetable oil. Below is a permanent slide from a wood section processed with ethanol and isopropanol, finally mounted in Euparal.
P1002184-Pano.jpg
P1002184-Pano.jpg (388.28 KiB) Viewed 6880 times

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#7 Post by Greg Howald » Mon Apr 04, 2022 2:14 pm

I agree with Microscopy is Fun. Find a different way to do this.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#8 Post by MicroBob » Mon Apr 04, 2022 3:12 pm

What functions do you expect from the clearing agent - remove paraffin?

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#9 Post by SuiGenerisBrewing » Mon Apr 04, 2022 3:36 pm

I'm surprised at the negativity in this thread to these results. Reasonable fixation/clearing of human biopsies and plant materials using safe, easily acquired materials is great for the home experimentalist (or for people looking to do this kind of experimentation in K12, or even in some undergrad labs). It opens up these procedures to people who may lack access to the traditional reagents, want cheaper options or a way to try a new form of processing without significant investment, or whom simply don't want those types of chemicals in their homes.

From a chemical perspective, the difference in using an oil versus xylene for clarification is minimal. The goal of clarification is to get rid of the dehydrating agent (alcohol), which tends to be incompatible with embedding agents. Alcohols absorb just as well into oils as xylene, and the refractive indices are similar. Honey instead of formalin is a bigger difference - formalin fixes by cross-links proteins, while honey acts by dehydration. In most cases this doesn't matter, outside of some specific specialized staining approaches. I'd be interested to see how an 8.2% solution of glucose or sucrose works, as that would be similar in sugar content to 1:10 diluted honey, but without the potential for pollen or other contaminants.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#10 Post by BramHuntingNematodes » Mon Apr 04, 2022 3:38 pm

Hobbyst46 wrote:
Mon Apr 04, 2022 10:24 am

The chemistry of the effect of formalin on tissues is not the same as the chemistry of the effect of alcohols, honey or oils. Formalin has been used for many generations. yes, it is toxic, carcinogenic and more. Yes, its smell is disgusting. Yes, it should be definitely avoided in home labs, where safety measures and facilities are limited. Xylene is hazardous though less than formalin. But for some purposes there are no replacements for the real stuff.
Moreover, any chemical that achieves what formalin does will likely be as toxic because what formalin does is antithetical to life.

I use it sparingly for botanical fixation, but the usual method of nematode preservation-- scalding the 'todes to death in hot formalin-- is rumored to have led to illness in many nematologists. Of course there is a difference between occupational exposure over a long period versus occasional hobbyist use but even once using this method seems like a bad idea, particularly without a fume hood.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#11 Post by SuiGenerisBrewing » Mon Apr 04, 2022 4:18 pm

BramHuntingNematodes wrote:
Mon Apr 04, 2022 3:38 pm
Hobbyst46 wrote:
Mon Apr 04, 2022 10:24 am

The chemistry of the effect of formalin on tissues is not the same as the chemistry of the effect of alcohols, honey or oils. Formalin has been used for many generations. yes, it is toxic, carcinogenic and more. Yes, its smell is disgusting. Yes, it should be definitely avoided in home labs, where safety measures and facilities are limited. Xylene is hazardous though less than formalin. But for some purposes there are no replacements for the real stuff.
Moreover, any chemical that achieves what formalin does will likely be as toxic because what formalin does is antithetical to life.

I use it sparingly for botanical fixation, but the usual method of nematode preservation-- scalding the 'todes to death in hot formalin-- is rumored to have led to illness in many nematologists. Of course there is a difference between occupational exposure over a long period versus occasional hobbyist use but even once using this method seems like a bad idea, particularly without a fume hood.
Have you tried METHAC (8:1 methanol:glacial acetic acid)? My colleague uses that for fixing agriculturally important nematodes with good success. Unlike formalin, it also has the advantage that it allows for DNA analyses to be performed afterwards.

On a semi-related note, formalin is aqueous formaldehyde, and formaldehyde (in small quantities) is required for life. It is part of the 1-carbon pathway needed to synthesize DNA and some amino acids. Obviously, in larger amounts its damned toxic.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#12 Post by BramHuntingNematodes » Mon Apr 04, 2022 4:28 pm

Oh yes, thank you, I'm quite familiar with combinations of acetic acid and alcohols.

Sure we might use some molecules of the stuff here and there in our bodies, but what we want formalin to do as a fixative, penetrate tissue and indiscriminately bind to all proteins, makes it an excellent all-purpose death agent on the microscopic level. Which again, any chemical that does that would be.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#13 Post by AquaLifefu » Mon Apr 04, 2022 6:46 pm

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668515/ Bio-Friendly Alternatives for Xylene – Carrot oil, Olive oil, Pine oil, Rose oil

From this research, the use of these oil cause less shrinkage, while pine oil actually show better results than xylene, they were place in hot air oven at 140F for 40 minutes for two cycles(correct me if I'm wrong but with that low of tempature, I would assume oven from home would be sufficient.).

I already have Eurpral and already using mixture of denatured alcohol to get my proper dehydration , I'm only putting up this discussion for those that would prefer a safer alternative and to see if anyone have any input in this since I have not seen any thread about this technique .

"Results
Among the 4 different oils used pine oil has similar colour, viscosity properties with that of xylene and is very economical as compared to xylene. Rose, Carrot and Pine oils are more viscous than xylene but when subjected to heat they showed equal penetration to that of xylene [Table/Fig-3]. Tissues cleared in different oils showed similar gross changes compared to that of xylene. Pine oil was superior in translucency and caused less shrinkage of tissue compared with that of xylene. On other hand carrot and rose oils showed less shrinkage of tissue compared to xylene. Translucency of the tissues cleared in carrot and rose oils was similar to tissues cleared in xylene [Table/Fig-4]. Tissue cellular architecture was preserved in all the sections cleared with the four different oils and a clear distinction was observed between nucleus and cytoplasm. The overall staining quality almost equivalent with that of xylene. "

Though, despite it benefits, what have not know is it long term stability as most of these research are pretty recent.


Results from. " Quest for Biofriendly Xylene Substitutes in Histopathology: A Comparative Study" using extra virgin olive oil and refined sunflower oil.


https://www.semanticscholar.org/paper/Q ... d9c9663b2b

Image


Image

"Results: Results showed both the oils had the ability to clear and deparaffinize tissues. Extra virgin olive oil was comparatively better, it maintained the tissue integrity, and the overall staining quality was also better when compared to RSO.

Conclusion: To conclude, extra virgin olive oil and RSO can be used as a biofriendly substitute to xylene in histopathological laboratory."

For those that interested in using this technique, this quote should be useful from the same study.

" Extra virgin olive oil and RSO do not have the ability to dissolve fat at room temperature, which xylene does; therefore, they were used at 30°C, 60°C, and 90°C as an increase in temperature causes the tissue fats to dissolve readily, ensuring proper clearing of tissues. Optimum results were obtained at 60°C. In addition to that increase in the temperature, increases the kinetic energy and the rate of dif f usion of oil molecules, thereby leading to decrease in viscosity. This ensured greater penetration of the extra virgin olive oil, RSO and faster removal of the dehydrating medium. Similarly, deparaffinization procedure was also tried at 30°C, 60°C, and 90°C, and the optimum results were obtained at 60°C. There were certain advantages noted while using extra virgin olive oil and RSO as clearing and deparaf f i nizing agents (Table 3). The density of extra virgin olive oil and RSO is closer to that of the density of animal fat, which allowed them to cause displacement of fat, rather than dissolution, as xylene does. Time taken for staining was comparatively less than the conventional procedure which is in accordance with the study by Premalatha et al., in 2013.6"
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#14 Post by Microscopy_is_fun » Mon Apr 04, 2022 7:25 pm

AquaLifefu wrote:
Mon Apr 04, 2022 6:46 pm
I'm only putting up this discussion for those that would prefer a safer alternative and to see if anyone have any input in this since I have not seen any thread about this technique .
Hi AquaLifefu,

first thanks for all your research on this topic and your input! Actually I would be happy to replace all this volatile and in some cases toxic solvents in microscopy.

That's why I looked into the papers which you referenced, and there I mostly got confused. Some of the authors where obviously using paraffin for embedding. After slicing the parraffin sections, the slices are fixed to the slide. Prior to staining, the paraffin needs to be removed, typically with xylene. In the references this step is not described, at least I couldn't find that. I assume the oil wouldn't do a good job here, because you will always have residues of oil (and possibly paraffin) on the slide. How did the authors solve that?

In the case of paraffine-free mounts you might have the same issue, meaning that some oil will finally end up in the mount. With volatile solvents, this is not a problem, since isoprop and xylene evaporate and are therefore less likely to leave residues in the final permanently mounted slide. The open point in my view is therefore: How do you get rid of the oil, once it has done its job?

My skepticism is therefore rather related to the incomplete descriptions in the cited articles, and less to the general approach, which would be great if it really worked.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#15 Post by AquaLifefu » Mon Apr 04, 2022 9:41 pm

Microscopy_is_fun wrote:
Mon Apr 04, 2022 7:25 pm
AquaLifefu wrote:
Mon Apr 04, 2022 6:46 pm
I'm only putting up this discussion for those that would prefer a safer alternative and to see if anyone have any input in this since I have not seen any thread about this technique .
Hi AquaLifefu,

first thanks for all your research on this topic and your input! Actually I would be happy to replace all this volatile and in some cases toxic solvents in microscopy.

That's why I looked into the papers which you referenced, and there I mostly got confused. Some of the authors where obviously using paraffin for embedding. After slicing the parraffin sections, the slices are fixed to the slide. Prior to staining, the paraffin needs to be removed, typically with xylene. In the references this step is not described, at least I couldn't find that. I assume the oil wouldn't do a good job here, because you will always have residues of oil (and possibly paraffin) on the slide. How did the authors solve that?

In the case of paraffine-free mounts you might have the same issue, meaning that some oil will finally end up in the mount. With volatile solvents, this is not a problem, since isoprop and xylene evaporate and are therefore less likely to leave residues in the final permanently mounted slide. The open point in my view is therefore: How do you get rid of the oil, once it has done its job?

My skepticism is therefore rather related to the incomplete descriptions in the cited articles, and less to the general approach, which would be great if it really worked.
Maybe someone else would answer this better but with this paper, this hopefully is the answer I found to both of your question.

https://www.arpapress.com/Volumes/JPCS/ ... S_8_02.pdf

This technique use bleach palm oil.

"group B tissues were dehydrated through ascending grades of ethanol (70%, 90%, 95%, absolute 1, absolute 2 and absolute 3) for 1 hour in each change. Dehydrate tissues were dealcoholized (cleared) by using two changes of bleached palm oil at 60C for 1 hour each. The cleared tissues were infiltrated in 2 changes of molten paraffin wax (wax 1 for 1 hour and wax 2 for 1 hour 30 minutes). Embedding was done in molten paraffin wax using plastic disposable cassettes and allowed to solidify before microtomy. Tissue blocks were all sectioned at 4 µm with a rotary microtome; sections were floated in a warm water bath and each section picked in pairs on albuminized glass slides.

Before staining, the sections were dewaxed in pre-warmed bleached palm oil at 60C for 15 minutes. The slides were stood upright for 1 minute to drain off excess oil and rinsed in 2 changes of 1.7% dish washing solution pre-warmed at 60C for 10 minutes each to degrease the sections before rinsing in water. Hydrated sections were stained in Cole’s haematoxylin solution for 10 minutes and rinsed in water. The sections were counterstained in 1% eosin solution for 1 minute, rinsed in water, air dried and mounted in DPX 10, 11..."

" In addition, the use of 1.7% dish washing soap solution at 60C for degreasing the sections after dewaxing in bleached palm oil contributed to clarity of staining observed. First, at 60C, the viscosity of bleached palm oil is reduced to 16.93 mPas from 77.19 mPas at 25C 18. This increased the fluidity of the bleached palm oil and allowed its easy emulsification and removal by the soap solution. During emulsification, the soap forms an interface (micelle) between the water and oil resulting in gradual dissolution of the oil into water 19.There was no observable changes attributed to 1.7% dish washing soap solution on the histology and quality of staining of the tissues since it has been used as a dewaxing solution at 90C to replace xylene during staining... "

From what I understand, and this is the simplest way as possible to do this from what I read in the article , instead of xylene, I would use two part of oil, first oil part at 60c for an hour in the oven then move the speciment to the second oil container also at 60c for another hour. I don't use paraffin so move to greasing step, let it stand vertically to drain off excess oil for about 2 minutes. Then submerged speciment in pre-warm 60c tempature mixture of 1.7% dishwashers soap and water for 10 minutes, then redo the dishwasher soap and water part again, then air dried. Correct me if I'm wrong or pharse it in a better way.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#16 Post by Microscopy_is_fun » Mon Apr 04, 2022 10:31 pm

AquaLifefu wrote:
Mon Apr 04, 2022 9:41 pm

Before staining, the sections were dewaxed in pre-warmed bleached palm oil at 60C for 15 minutes. The slides were stood upright for 1 minute to drain off excess oil and rinsed in 2 changes of 1.7% dish washing solution pre-warmed at 60C for 10 minutes each to degrease the sections before rinsing in water.

From what I understand, and this is the simplest way as possible to do this from what I read in the article , instead of xylene, I would use two part of oil, first oil part at 60c for an hour in the oven then move the speciment to the second oil container also at 60c for another hour. I don't use paraffin so move to greasing step, let it stand vertically to drain off excess oil for about 2 minutes. Then submerged speciment in pre-warm 60c tempature mixture of 1.7% dishwashers soap and water for 10 minutes, then redo the dishwasher soap and water part again, then air dried.
Using hot water and soap might do the job, that could be worth a try. I am mostly interested in botanical samples, where you have tiny bits and pieces on the slide. Wether they survive the harsh treatment at 60°C is probably the open question to be answered.

At the moment I play around with the standard xylene-paraffin-approach, to be honest with limited success. Once I suceed with this standard-procedure, I might have a look into the "oil-business". Actually I am not too much worried about health issues, but the hazardous waste associated with xylene, paraffin and methylbenzoate, plus the hassle in buying those chemicals as private person makes alternatives worth a try.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#17 Post by AquaLifefu » Mon Apr 04, 2022 10:37 pm

Microscopy_is_fun wrote:
Mon Apr 04, 2022 10:31 pm
AquaLifefu wrote:
Mon Apr 04, 2022 9:41 pm

Before staining, the sections were dewaxed in pre-warmed bleached palm oil at 60C for 15 minutes. The slides were stood upright for 1 minute to drain off excess oil and rinsed in 2 changes of 1.7% dish washing solution pre-warmed at 60C for 10 minutes each to degrease the sections before rinsing in water.

From what I understand, and this is the simplest way as possible to do this from what I read in the article , instead of xylene, I would use two part of oil, first oil part at 60c for an hour in the oven then move the speciment to the second oil container also at 60c for another hour. I don't use paraffin so move to greasing step, let it stand vertically to drain off excess oil for about 2 minutes. Then submerged speciment in pre-warm 60c tempature mixture of 1.7% dishwashers soap and water for 10 minutes, then redo the dishwasher soap and water qpart again, then air dried.
Using hot water and soap might do the job, that could be worth a try. I am mostly interested in botanical samples, where you have tiny bits and pieces on the slide. Wether they survive the harsh treatment at 60°C is probably the open question to be answered.

At the moment I play around with the standard xylene-paraffin-approach, to be honest with limited success. Once I suceed with this standard-procedure, I might have a look into the "oil-business". Actually I am not too much worried about health issues, but the hazardous waste associated with xylene, paraffin and methylbenzoate, plus the hassle in buying those chemicals as private person makes alternatives worth a try.
Definitely post it here or pm me how your result with your bonatical sample with the oil. I'm currently trying it out with my spider sample.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#18 Post by Microscopy_is_fun » Tue Apr 05, 2022 6:43 am

AquaLifefu wrote:
Mon Apr 04, 2022 10:37 pm
I'm currently trying it out with my spider sample.
Please let us know, how your "fried spider" finally turned out!

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#19 Post by Alexander » Tue Apr 05, 2022 6:59 am

Microscopy_is_fun wrote:
Mon Apr 04, 2022 7:25 pm

That's why I looked into the papers which you referenced, and there I mostly got confused. Some of the authors where obviously using paraffin for embedding. After slicing the parraffin sections, the slices are fixed to the slide. Prior to staining, the paraffin needs to be removed, typically with xylene. In the references this step is not described, at least I couldn't find that. I assume the oil wouldn't do a good job here, because you will always have residues of oil (and possibly paraffin) on the slide. How did the authors solve that?
Xelene may be substituted by warm (120 - 140° F) isopropanol. At this temperature isopropanol easily dissolves paraffin.

There is an issue with Euparal and all the named oils as alternatives for xylene not yet mentioned. All this bio-friendly substitutes feature significant auto-fluorescence. If ones does fluorescence microscopy as I do, this is very disturbing. The only resin based mounting media free of autofluorescence I ever found on the market is Eukitt which is xylene based. There is no way for me to avoid xylene.

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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#20 Post by Microscopy_is_fun » Tue Apr 05, 2022 7:55 am

Alexander wrote:
Tue Apr 05, 2022 6:59 am
Xelene may be substituted by warm (120 - 140° F) isopropanol. At this temperature isopropanol easily dissolves paraffin.

There is an issue with Euparal and all the named oils as alternatives for xylene not yet mentioned. All this bio-friendly substitutes feature significant auto-fluorescence.
I tried the isopropanol-paraffin-route recently. It turned out to become a big mess, since the warm isoprop partially evaporates and takes some of the paraffin with it. I had paraffin residues all over the place. I therefore still prefer the xylene route so far.

About the autofluorescence of Euparal you mention a valid point. Permanent slides for fluorescence are out of the scope when using Euparal. In my case, I use only fresh samples for fluorescence, embedded in glycerol. Due to photobleaching, permanent mounts are not in my focus anyway.

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AquaLifefu
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#21 Post by AquaLifefu » Wed Apr 06, 2022 8:24 pm

Microscopy_is_fun wrote:
Tue Apr 05, 2022 6:43 am
AquaLifefu wrote:
Mon Apr 04, 2022 10:37 pm
I'm currently trying it out with my spider sample.
Please let us know, how your "fried spider" finally turned out!
Apparently hempsteadhalide send me hoyer instead of eurpral, i noticed that too late so not sure how it gonns turn out. My oven lowest is 170f so end up doing that, my hot water only do up to 120f, i thought I clean it well enough but the whole slide have a lot of oil stain afterward, gonna try it later with another speciment with 140f hot water and more quantities of soap to see if it would be better.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#22 Post by AquaLifefu » Fri Apr 15, 2022 3:49 am

Dishsoap and lemon water as Deparaffinizing.

https://www.ncbi.nlm.nih.gov/pmc/articl ... ort=reader

This seem to be easier to do and less clean up compare to the oil.
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Re: Coconut oil(vegetables oil) as clearing agent alternative to Xylene.

#23 Post by MicroBob » Fri Apr 15, 2022 8:37 am

I don't know whether this has already been mentioned, but there is Histoclear available as a Xylene substitute. I think user mrsonchus has mentioned using is successfully. It probably is based on orange peel oil so there is room for experimentation. I use orange peel oil to wipe away paraffin redidues and it dissolves xylene at room temperature.

The methods with with dishwashing solution and lemon water don't seem very promising to be honest. The paper shows that it generally works but I'm fairly sure that no one would be satisfied enough to switch over to it.

I use xylene in tiny amounts in closed containers. Xylene is part of paints and thinners that are sold in ordinary hardware stores. Filling ut the tank of a car probably brings more contact to harmful substances than using xylene in a well controlled manner. So I don't think it is necessary to spend much energy on avoiding it completely.

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